Laminitis is a common and often life-threatening condition in the horse. The condition is either at the top or within the top three priorities for every major funding agency in North America. Irrespective of the initiating cause, the end result is mechanical failure of the hoof due to varying degrees of separation of the insensitive part of the hoof from the underlying sensitive tissues. For over three decades, North American researchers have focused on unraveling a complex series of vascular events believing a state of under-perfusion exists during the development of the disease. Interestingly, these vascular events do not develop until after the onset of clinical lameness leaving many to question whether they are simply a response to the mechanical disruption of the hoof, rather than a cause of the condition. In fact, a recent report shows an increase in sub-laminar blood flow during the prodomal stages of experimentally induced laminitis (Pollitt et al., 1998).
Laminitis can be induced using a carbohydrate over-ingestion (CHO) model (Garner et al., 1975) and pre-treatment with virginiamycin will prevent the development of clinical laminitis suggesting microbial involvement in the development of the disease (Rowe et al., 1994). The original CHO model is associated with unacceptable incidences of colic and mortality and has been replaced with an oligofructose model (Pollitt et al., 2003). Using this model, an overgrowth of Streptococcus species in the hindgut has been demonstrated (Milinovich et al., 2006). Ex vivo studies of the hoof have confirmed that matrix metalloproteinases (MMP) 2 and 9 are activated when hoof explants are exposed to the Streptococcus bovis exotoxin. The cysteine protease, Streptococcus pyrogenic exotoxin B (SpeB), also had a direct effect on the tissue. Both treatments resulted in dissolution of the basement membrane and separation of the hoof wall proper from the underlying sensitive tissues when mechanical traction is applied across the lamina (Mungall et al., 2001). The basement membrane failure is believed to result from the cleavage of the protein laminin which is an essential component of the cytoskeletal system that suspends the coffin bone within the hoof proper (French et al., 2004). Histologically, the lesion is identical to that observed in clinical manifestations of the condition (Mungall et al., 2001).
It is known that exposure of hoof explants to Streptococcus exotoxins results in local activation of MMPs and that MMPs are up-regulated in hoof explants from clinically affected horses. However, it is not known whether this up-regulation is limited to the foot or whether exotoxin is present in the hoof of clinically affected horses.
There is considerable evidence supporting a bacterial contribution in the pathogenesis of laminitis (Rowe et al., 1994; Milinovich et al., 2006; Mungall et al., 2001). Studies in mice and rabbits have shown antibodies to SpeB effectively inhibit protease activity and improve survival of mice challenged with intraperitoneal injections of Streptococcus pyogenes (Kapur et al. 1994). Streptococcus exotoxins SpeB and bacterial M4 family thermolysins are highly conserved (Kapur et all 1993; Adekoya and Sylte, 2009) and have been implicated in the development of laminitis (Mungall et al., 2001). Continuous cryotherapy is well tolerated and can effectively limit the hoof damage associated with the development of laminitis in horses undergoing oligofructose induced laminitis (Kapur et al., 1994; Kapur et al., 1993; Pollitt et al., 2004; van Eps et al., 2004). The observed protective effect may be a result of reduced delivery of laminitis “trigger factors” or possibly related to expected reduction of enzymatic activities due to reduced temperature (van Eps et al., 2009a). Irrespective of the mode of protection, clinical laminitis can be prevented by standing horses in knee deep ice-water for 72 hours during the oligofructose induction protocol. Furthermore, any histologic remnants of acute laminitis resolve within 7 days (van Eps et al., 2009b).
The primary focus of Streptococcal immunoprophylaxis has been M protein. Unfortunately, there is extensive variation between serotypes and concern that autoimmune responses can occur. Vaccination of mice against SpeB reduced morbitity and mortality due to a streptococcal infection model (Kapur et al. 1994). Antibodies raised against the 40 kDa precursor form of SpeB were not as effective as those raised against the 28 kDa cleaved form (Matsuka et al. 1999).
Mungall et al. (2001) discloses a possible link between laminitis and bacterial proteins, including thermolysin and SpeB. Mungall et al. further discloses that Streptococcus bovis is a key bacterium responsible for fermenting starch in the equine hindgut.
Sudheesh et al. (2007) discloses that the thermolysin protein of Flavobacterium psychrophilum is antigenic and that this protein may be used for targeted vaccine development against bacterial coldwater disease and rainbow trout fry syndrome in fish.
European Patent No. EP 0781340 discloses a vaccine against a Streptococcal exotoxin B (SpeB) protein comprising at least one amino acid substitution at Cys192 or His340, which disrupts cysteine protease activity. The vaccine was produced by inoculating mice with purified SpeB subcutaneously followed by intraperitoneal boost.
WO/1996/008569 discloses a vaccine against a Streptococcal (specifically S. pyogenes) extracellular cysteine protease (SpeB), or a fragment thereof.
U.S. Pat. No. 6,287,555 discloses a vaccine against Streptococcus bovis, using live cells, attenuated cells, killed whole cells, or cell lysate as an inoculant, for the purpose of protecting animals, including horses, from the effects of overproduction of acid and further discloses that overproduction of acid causes a variety of conditions, including laminitis.
U.S. Pat. No. 7,011,826 discloses the use of a vaccine comprising a live or dead Streptococcus strain, selected from specific Streptococcus strains including S. bovis SbR1 and S. equinus SER1, to protect against acidosis and further discloses that acid accumulation in the gut can result in laminitis in horses.
U.S. Pat. No. 5,641,492 discloses a vaccine for protecting horses and cattle against endotoxin-associated diseases, including laminitis. The vaccine comprises a killed suspension of a bacterial mutant, particularly a Salmonella mutant, which lacks an 0-carbohydrate side chain.